PROJECT SUMMARY Viruses are the most abundant biological entities on Earth and have been one of the greatest threats to human health. Despite their ubiquity and impact, less than 0.01% of viruses are identified and well-characterized. Recent study showed that there are about 108 different viral species while only a few hundred of them have been studied. One major roadblock to isolating and studying viruses is the inability to grow them in culture. In fact, most of the known viruses cannot be grown in the laboratory. Another limiting factor in the study of viruses is the relatively low abundance of virus particles present in complex specimens such as human tissue or bodily fluids. This application seeks support to build new platforms enabling genome sequencing of single virions and isolation of extremely rare virions. Single virion sequencing will eliminate the need for establishing cultivable virus-host system and enable genome sequencing of uncultivable viruses directly from environmental samples. The new platform will integrate metagenomics, drop-based microfluidics, and up-to-date molecular biology techniques. Drop-based microfluidics is a technique that utilizes micron size channels to precisely control and manipulate small volumes of fluids separated by immiscible phase at a very high throughput. Within an hour, a microflulidic drop maker generates millions of picoliter-size test tubes that function as reaction vessels. In addition, individual droplets can be merged to add reagents, split to subtract droplet contents, or passed through a fluorescence detection system to detect droplets that are positive to the assays at the rate of ~ 1000/s. Droplet microfluidic techniques will be used to encapsulate single virions into micron-sized drops, perform single virus assays, and select target virions at an extremely high throughput. Metagenomics is the study of genetic materials recovered directly from environmental samples and enables researchers to sample all DNA fragments in a given sample. Metagenomic analysis will be used to identify the partial sequence of a target virus, and these sequences will be used to detect and isolate the target virus from a complex biological mixture. The first platform we propose will enable isolation and genome sequencing of a single selected virion. Using the platform, we will identify the genome sequence of novel viruses that are potentially human pathogens, African swine fever-like virus (ASFLV) and human rhino-like virus (HRLV). The second platform we propose will enable parallelized sequence analysis of single intact virions. We will use this platform to perform high throughput sequencing of single virions that are randomly sampled from untreated wastewater. Recent studies indicate that the vast majority of virions in wastewater are novel viruses; therefore, high-throughput genome sequencing of single virions in wastewater will reveal the genome sequence of diverse novel viruses. The successful merging of microfluidics and metagenomics towards the characterization of viral genomes will not only help identifying novel viruses that can be possible threats to humans but also open new avenues for the investigation of virus diversity, evolution, and adaptation.